This invention relates generally to diagnostic immunoassays for medical applications, and more particularly to methods for rapidly determining whether a person has suffered an acute myocardial infarction.
Acute Myocardial Infarction (AMI), commonly referred to as a heart attack, is a disorder in which a region of the heart muscle is damaged due to an inadequate supply of oxygen thereto. For instance, a clot in a coronary artery that blocks the supply of oxygen to the particular region of the heart and results in death or damage to the cells in this region can cause an AMI. Typically, the AMI occurs as a result of a coronary artery that has been narrowed due to the effects of arteriosclerosis. The damaged tissue causes a permanent loss of contraction of this portion of the heart muscle.
There is a great desire for early determination as to whether AMI is occurring for patients that present themselves at an emergency room of a hospital. A significant number of patients complain from chest pain and/or other symptoms concomitant with AMI (Heden et al., Circulation, 96: 1798-802 (1997)). This early determination allows hospital personnel to determine which patients can be sent home and which patients have suffered an AMI. For patients who have suffered an AMI, thrombolytic therapy should be started as soon as possible after the AMI for maximum benefit. Unfortunately, a high percentage of patients who present with chest pains are admitted to the intensive care unit or telemetry ward of the hospital. In addition, a small percentage of patients that are sent home after the clinicians determined such patients did not suffer an AMI, shortly thereafter are admitted to the hospital as having suffered an AMI. These problems are due to traditional diagnostic regiments lacking sufficient sensitivity in a large portion of the cases presented.
Currently, electrocardiographs (ECG) are used in concert with blood serum protein elevations to diagnose the infarction. It has been shown that the diagnostic sensitivity of an ECG is approximately 50% in determining myocardial damage (Gibler et al., Ann Emerg Med 19:1359-66 (1990)). The number of individuals who have been discharged from emergency departments provides further evidence of the need for a more efficient method of AMI detection. It has been indicated that approximately 25% of the patients sent home with an acute myocardial infarction had ST elevations that were misjudged or overlooked by the physician (McCarthy et al., Ann Emerg Med 22:579-82 (1993)). In addition, an estimated 80% of the patients admitted to the coronary care unit for suspected acute myocardial infarction are discharged without having this diagnosis confirmed (Puleo et al., N Engl J Med, 331:561-66 (1994); Roberts & Kleiman, Circulation, 89(2):872-81 (1994)). More importantly, 2-8% of the 2 million individuals that were not admitted developed AMIs, resulting in added injury to the individual and malpractice actions taken against the institution (Hudson et al., Clinica Chimica Acta, 284(2):223-37 (1999)).
Due to the increasing number of AMI patients accompanied with a difficulty in diagnosing the anomaly, a major push to develop innovative approaches to tackle the recognition of myocardial infarctions has been noticed. Most of these advances utilize immunoassays to decrease the time of AMI detection, granting the patient a better chance of survival (Hudson et al., Clinica Chimica Acta 284:223-37 (1999)).
The release of specific cardiac proteins from the injured cardiac tissue into the patient's bloodstream has become an important parameter for the diagnosis of AMI. A proper protein marker to use in such an assay is one that is present in measurable concentrations in the early AMI onset stage as well as having high clinical sensitivity and specificity (De Winter et al., Circulation, 92(12):3401-07. Several proteins or markers can result from the cardiac tissue injury. Myoglobin, creatine kinase, fatty acid-binding protein (FACB), cardiac specific troponins, and glycogen phosphorylase (AP) are released into the blood stream immediately after the cardiac injury. The measurement of these markers has become an increasingly important for the diagnosis and sizing of AMI. Depending on the differences in their concentration in the blood stream, these markers are used both for diagnosis, by detecting the presence of increase concentration of these proteins in patients who have AMI signs, and sizing of an AMI, by taking serial measurements of these markers to estimate the extent of damage (Adams et al., Circulation, 88(2):750-63 (1993)).
An ideal marker that could be used to detect myocardial injury would possess a number of characteristics. It must have high protein concentrations within the myocardium, while being present in low concentrations in noncardiac tissue. It also should be rapidly released after cardiac injury, persists in plasma, and allow the development of accurate and rapid assays (Adams, Clinica Chimica Acta 284(2):127-34 (1999)).
The CK-MB isoenzyme (86 kDa) is one of the most common markers used for the evaluation of patients with suspected AMI (Achar et al., Am. Fam. Physician 72(1):119-26 (2005)). Damaged myocardial tissue releases CK-MB in a characteristic fashion following the injury, allowing clinicians to often determine the timing and extent of an AMI, with the concentration level of CK-MB normally being 3 U/L and peaking at over 25 U/L 16-20 hours after infarction. Unfortunately, CK-MB is insufficiently sensitive during the early phases of an AMI, with an increase in concentration also present during massive skeletal muscle damage. The late peak of CK-MB concentration has led to its primary use in confirming an AMI at 24 hours post-injury (Id.; Cubrilo-Turek et al. Acta Med Croatica 58(5):381-8 (2004); Gibler et al., Ann Emerg Med 19:1359-66 (1990)). Recently, the use of the two CK-MB subforms (CK-MB 1 and CK-MB 2) has been proposed for a more accurate diagnosis of early AMI. CK-MB 2 is found in myocardial tissue, and upon tissue destruction is released into the blood as the more negatively charged subform, CK-MB 1. A level of CK-MB>1 U/L and a ratio of CK-MB 2 to CK-MB1>1.5 has been reported to have a better sensitivity within the first six hours post-AMI compared to CK-MB alone (Penttila et al., Clin Biochem 35(8):647-53 (2002); Lin et al., Clin Chem 50(2):333-38 (2004)). Troponin is a protein also found in the skeletal and cardiac muscle that has become a standard marker for the diagnosis of AMI (Achar et al., Am Fam Physician 72(1):119-26 (2005); Berroeta et al., Ann Fr Anesth Reanim, (2005)). Used in conjunction with CK-MB, troponin can detect not only minor myocellular necrosis but also can be used as an indicator for patients at risk for ACS [Penttila et al., Clin Biochem 35(8):647-53 (2002); Lin et al., Clin Chem 50(2):333-38 (2004); Berroeta et al., Ann Fr Anesth Reanim, 2005)). Troponin entails three forms (C, I, and T) which along with actin and myosin regulate muscle contraction. While troponin C is identical in cardiac and skeletal muscles, troponins I and T are site specific. Therefore, a rise in cardiac specific troponin I and T is suggestive for myocardial damage. Both forms rise within the first 3-4 h of the injury, peak generally within 24 h, and remain elevated for 10-14 days, therefore qualifying as late markers for a recent AMI in patients who do not receive an immediate medical assistance. Although troponin I and T have similar sensitivity and specificity for the detection of myocardial infarction, troponin T may be elevated under other pathological conditions such as renal failure or polymyositis. Recently, the use of troponin I over the T isoform in the diagnosis of ACS and AMI has been reported (e.g., Eisenman et al., Singapore Med. J 46(7):325-27 (2005). Moreover, troponin I alone has been shown to be a better marker for risk stratification in patients with chest pain than when combined with CK-MB and myoglobin Qggers et al., Am Heart J. 148(4):574-81 (2004); Eggers et al., Coron Artery Dis 16(5):315-19 (2005)). Recent studies have shown that FABP and myoglobin can be used to detect a myocardial injury sooner after an infarction than CK-MB or troponin T (Achar et al., Am Fam Physician 72(1):119-26 (2005); Ishii et al., Clin Chem (2005); Chen et al., J. Huazhong Univ Sci Technolog Med Sci 24(5):449-51, 459 (2004); Sallach et al., Am J. Cardiol 94(7):864-67 (2004)). Myoglobin (17 kDa) and FABP (15 kDa) are two small cardiac proteins that show elevated serum levels soon after the infarction, significantly increasing their concentration within 2 hours after infarction and peeking 4-6 hours after an infarction. The concentrations of myoglobin and FABP in plasma rise from about 32 μg/L and 3 μg/L respectively to over 200 μg/L for myoglobin and 100 μg/L for FABP often under 5 hours after an AMI (Van Nieuwenhoven et al., Circulation 2848-54 (1995); Wodzig et al., Eur. J Clin Chem Clin Biochem 71:135-40 (1997)). Myoglobin is a heme protein that participates in the oxygen transport in the muscle, hence any severe destruction of striated muscle may result in significant elevations of its plasma concentration. Although myoglobin has a low cardiac specificity, it has a high sensitivity for myocardial injury that makes is a useful marker for AMI when used in parallel with other cardiac markers. Recent studies show that the combined measurement of myoglobin and FABP in plasma allows the discrimination between myocardial and skeletal muscle injury with the ratios for these markers differing between heart (MYO/FABP ratio 4:5) and skeletal muscle (MYO/FABP ratio 20:70, depending on muscle type). Alternately, combined measurements of myoglobin concentrations with skeletal muscle specific markers by supplementary tests such as CK-MB, cardiac specific troponins, or carbonic anhydrase III can also be used to differentiate between myocardial and skeletal muscle damage. A potential drawback is that both markers, FABP and myoglobin, unlike other cardiac markers, are eliminated from plasma by renal clearance, which means that measurements could be inaccurate in patients with chronic renal failure. In these cases, cardiac markers concentrations should be interpreted in conjunction with the individual estimated renal clearance rates of the patients. Although both markers lack cardiac sensitivity, myoglobin has been used more often as an early AMI marker. Despite recent studies that indicate cardiac FABP may provide information superior to other markers in the early onset of ACS (Ishii et al., Clin Chem (2005); Chen et al., J. Huazhong Univ Sci Technolog Med Sci 24(5):449-51, 459 (2004)), its use in the diagnosis of myocardial injury is not unanimously accepted.
Development of a cost effective, simple and efficient assay determining whether an AMI has taken place would greatly assist health care providers in timely and accurately diagnosing AMI suspected patients. It would therefore be desirable to provide improved methods and assay compositions for AMI detection. It would also be desirable to provide better methods and assays for measuring myoglobin, as well as other assayable antigens.